When endogenous repair fails, as is often the case with dense fibrous musculoskeletal tissues (like the knee meniscus), novel strategies and enabling technologies must be developed to enhance tissue regeneration. While numerous tissue engineering therapies have been developed for the engineering of fiber-reinforced tissues, one persistent limitation is the ability to fabricate scaffolds that maintain structural integrity and direct appropriate tissue architecture, while simultaneously promoting cellular infiltration throughout the regeneration period. In this proposal, we address these limitations with the production of a novel multi-polymer composite nanofibrous scaffold that provides a 3-dimensional micropattern for neo-tissue formation. These scaffolds, with fiber diameters on the order of the native extracellular matrix, can be produced with defined fiber anisotropies that mimic the structural arrangement of fiber-reinforced tissues. Further, by introducing flexibility in polymer properties via a library of photocrosslinkable macromers that exhibit a range of mechanical and degradation properties when polymerized, we propose to tailor temporal pore formation within the scaffold through the controlled degradation of individual polymer components. We hypothesize that these nanofibrous multipolymer photocrosslinked meshes will have controlled mechanical properties reflective of the stiffest and slowest degrading component and show a time dependent increase in void space while maintaining their overall mechanical properties. Further, we hypothesize that the controlled increase in void space within these scaffolds, via the erosion of sacrificial elements, will promote cellular infiltration into the multi-polymer mesh and will result in a more uniform functional tissue structure. Additionally, as the mechanical environment of the meniscus is paramount in its maturation and homeostasis, we hypothesize that tailored mechanical preconditioning regimens will likewise promote functional maturation of infiltrated meniscus constructs. To this end, a novel dynamic loading bioreactor that applies compression and tension is developed to promote tissue maturation. The first Aim of this proposal is to develop technology to electrospin multi-component nanofibrous scaffolds and compare properties to the native tissue using a predictive fiber-reinforced composite model. In the second Aim, the interaction and infiltration of cells and ultimate mechanical properties will be explored in single- and tri-polymer cell-laden nanofibrous scaffolds. The third Aim involves the development of a bioreactor for pre-conditioning scaffolds and evaluating the impact of mechanical stimulation on tissue formation in the short and long term. If successful, this innovative approach will provide several new enabling technologies for the functional regeneration of damaged fiber-reinforced musculoskeletal tissues. Public Health Relevance Statement (provided by applicant): This project develops a novel multi-polymer nanofiber fabrication system to exert control over polymer chemistry and overall scaffold mechanics and degradation with time to improve cellular infiltration and uniform tissue deposition in fibrous tissue-engineered constructs. If successful, this approach would surmount a major hurdle in the tissue engineering of dense structures of the musculoskeletal system and provide a mechanically functional, structurally anisotropic 3D micro-pattern for directed neo-tissue formation while promoting full cellular colonization and eventual replacement of the polymer structure after complete dissolution. This innovative approach, coupling scaffold fabrication, mechanical loading, and in vivo assessments, will aid in the development of tissue engineered therapies for fiber-reinforced musculoskeletal tissues such as the knee meniscus that otherwise fail to heal and have few clinically viable repair strategies.